Nociceptin-based analgesics

ABSTRACT

The invention relates to a family of hexapeptide compounds exhibiting activity with regard to the ORL-1 receptor. The compounds share a general formula of Arg-Tyr-Tyr-Arg-Trp-Arg, and may be constructed having modifications or substitutions at any position, and may include modifications of the amino- and carboxy-termini of the hexapeptide. These compounds include agents exhibiting agonist activity and antagonist activity when exposed to the human ORL-1 receptor. As such, the hexapeptides may be useful as analgesics, anxiolytics, diuretics, and anti-cancer agents.

RELATED APPLICATIONS

[0001] This application is related to and claims the benefit of U.S.Provisional Patent Application No. 60/327,888, filed Oct. 9, 2001, ofAmrit K. Judd entitled “Development of Nociceptin-Based Analgesics,”which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to analgesic compounds targeted tothe ORL1 receptor. More specifically, the present invention relates toagonist and antagonist compounds targeted to the ORL1 receptor andmethods for their use.

[0004] 2. Description of Related Art

[0005] It has been estimated that as much as 30% of the population ofthe industrialized countries of the world suffers from some degree ofchronic pain. Many individuals suffering from chronic pain are forced toincur significant direct medical and pharmaceutical expenses. Suchindividuals often also suffer losses in income and productivity. In theUnited States, it is estimated that the combined value of these lossesand costs is in excess of $50 billion annually.

[0006] To address the pain experienced by these millions of individuals,a large industry has developed to provide medications for controllingpain. The market for these analgesic drugs, broadly classified asnonsteroidal anti-inflammatory drugs and opiates, has become the largestin the world, with sales revenues estimated to be as high as 4.4 billionin 1996.

[0007] The analgesics sold in this market are among the most widely usedcompounds in the history of medicine. These products come in many forms,and include natural compounds and synthetic compounds which work safelyand with varying degrees of effectiveness to ease the impact of pain onan individual. Many of the compounds used for severe pain are opiatessuch as morphine and synthetic morphine analogs. These compounds havebecome widely used and understood.

[0008] Despite their effectiveness against severe pain, opiate compoundsare administered with caution, and their use is often restricted torelatively short time periods as a result of the side-effects andlimitations often connected with their use. Many patients experiencegastrointestinal side-effects which limit their ability to tolerate themedication for long periods of time. Other patients develop tolerance toopiates over time, resulting in diminished relief when opiate use isprolonged. Additionally, opiates carry a high potential for addiction,thus further endangering a patient. Accordingly, a need exists foreffective, nonaddicting analgesic compounds which cause few, if any,undesirable side effects.

[0009] Opiates include a large class of compounds that act on opiatereceptors, thus modulating the pain response in an individual. Threemain subclasses of opiate receptors have been identified through bindingstudies, in vitro and in vivo pharmacology, autoradiography, andreceptor cloning. Evans et al., Science, 258:1952-1955 (1992); Kiefferet al., Proc. Natl. Acad. Sci. USA, 89:12048-12052 (1992); Chen et al,Mol. Pharmacol., 44:8-12 (1993); Wang et al., Proc. Natl. Acad. Sci.USA, 90:10230-10234 (1993); and Yasuda et al., Proc. Natl. Acad. Sci.USA, 90:16736-16740 (1993). The μ, δ, and κ receptors are the apparentreceptors acted upon by common opiate drugs.

[0010] During research aimed at characterizing opioid receptors, aclosely-related orphan receptor eventually designated opioid receptorlike 1 (“ORL1”) was identified. Mollereau et al., FEBS Lett., 341:33-38(1994); Wang et al., FEBS Lett., 348:75-79 (1994); Bunzow et al., FEBSLett., 347:284-288 (1994). Despite levels of homology with the μ, δ, andκ receptors similar to their own homology to each other, the ORL1receptor failed to bind opiate compounds with high affinity. Althoughetorphine and some dynorphin gene products do produce a 1000-foldhigher-than-expected response when exposed to ORL1, ORL1's failure tobind with other opiates demonstrates that though ORL1 is in the opiatereceptor family, it is not a true opiate receptor. Zhang and Yu, J.Biol. Chem., 270:22772-22776 (1995).

[0011] In 1995, an endogenous ligand for ORL1 was sequenced. Meunier etal., Nature, 377:532-555 (1995). The peptide ligand, called nociceptin,is a 17-amino-acid peptide with a sequence resembling that of someopioid peptides, including dynorphin. Nociceptin was shown to inhibitcAMP accumulation in CHO cells transfected with ORL1, while causing nochange in non-transfected parent cells.

[0012] In other studies, nociceptin showed low affinity for the μ, δ,and κ opioid receptors. Gintzler et al., Eur. J. Pharmacol., 325:29-34(1997). Nociceptin also stimulates [³⁵S]GTPγS binding in transfectedcells and inhibits electrically-induced contractions in mouse vasdeferens (MVD); and, to a lesser extent, in guinea pig ileum (GPI).Berzetei-Gurske et al., Eur. J. Pharmacol., 302:R1-R2 (1996). Furtherstudies showed that intracerebroventricular injections of nociceptindecreases hot plate escape jumping latency and a decrease in tail flicklatency in mice. Meunier et al., Nature, 377:532-555 (1995); andReinscheid et al., Science, 270:792-794 (1995).

[0013] Intrathecal administration of nociceptin also shows promisinguse. When administered to the spinal cord in the presence of morphine,the action of morphine is inhibited, and further, nociceptin hasanalgesic action in tail flick testing in mice and also increasesmorphine analgesia. Tian et al., Br. J. Pharmacol., 120:676-680 (1997).Nociceptin has also been shown to be involved in peripheral analgesia,inhibiting formalin pain when administered intrathecally. Yamamoto etal., Neutrosci., 81:249-254 (1997). It similarly acts analgesically whenadministered to rats in a hot plate test of rats with a chronicconstriction injury, a model of neuropathic pain. Yamamoto et al.,Neurosci. Lett., 224:107-110 (1997). Similar function was observed inmodels of chronic pain, and in diabetic mice. Kamei et al., Eur. J.Pharmacol., 370:109-116 (1999).

[0014] The results of these and other tests suggest that agonists of thenociceptin receptor may prove useful as non-opioid analgesics,potentially useful with neuropathic pain. Additionally, the resultssuggest that antagonists of the nociceptin receptor may likely exhibitanti-anxiety properties. Unfortunately, however, nociceptin, the naturalligand for ORL1, is difficult to administer to a patient, and onceadministered, nociceptin is very susceptible to the action of proteases.Accordingly, a need exists for compounds which act as agonists andantagonists of the ORL1 receptor that are more easily administered to apatient, and that are resistant to protease activity.

[0015] In more recent years, the pain medication market has expandedvery rapidly with the entry of COX-II inhibitors for use with arthriticpain. The novel NSAID medications Vioxx® made by Merck Inc., andCelebrex® made by Pfizer/Pharmacia have garnered huge popularity andwidespread use in combating pain. The sales of Celebrex® in 2001 alonewere 3.1 billion dollars. These drugs have been shown to avoid some ofthe gastrointestinal problems of traditional NSAIDS while providing goodrelief to patients. Some researchers remain concerned about theprospective cardiovascular side-effects of these drugs, however.Additionally, as with opioid medications, these NSAIDS are generallyineffective against neuropathic pain. Neuropathic pain is a conditionoften thought to stem from damage to nerves, and is often found indiabetic patients. As II diabetes levels continue to swell in the UnitedStates, it becomes obvious that a need exists for novel compounds whichare effective against neuropathic pain which are not found in thecurrent analgesic market.

[0016] Most known analgesic compounds are agonists of at least one of agroup of opioid receptors. These compounds bind to the receptor,stimulating pain relief. Other known compounds share a similarstructure, but merely compete for binding with agonist compounds. Thesecompetitive compounds are referred to as antagonists. Antagonistcompounds often exhibit anxiety-relieving, or “anxiolytic” propertieswhen administered to a patient. The high incidence of anxiety disorderssuggests that it would be a benefit to characterize novel anxiolyticcompounds.

[0017] Finally, recent research has shown that some compounds currentlyused for their analgesic properties also exhibit anti-cancer activity.Despite recent advances in medical technology and breakthroughs inmolecular medicine, cancer remains a difficult disease to treat. As aresult, any novel compound with anti-cancer properties is a welcomedimprovement in the art.

[0018] Thus, it would be an improvement in the art to provide compoundsincluding agonists and antagonists of the ORL1 receptor. Further, itwould be a benefit to provide novel compounds for use as analgesics.Similarly, it would be an improvement in the art to provide novelcompounds for use as anxiolytic agents. It would also be an improvementto provide novel compounds with anti-cancer properties.

[0019] Such compounds and methods of their use are disclosed herein.

SUMMARY OF THE INVENTION

[0020] The present invention has been developed in response to thepresent state of the art, and in particular, in response to the problemsand needs in the art that have not yet been fully solved by currentlyavailable analgesic compounds. Thus, the present invention providescompounds such as nociceptin agonists and antagonists for use asanalgesic agents.

[0021] The invention includes a family of hexapeptide nociceptin analogsincluding compounds exhibiting full agonist activity and full antagonistactivity. The antagonist peptide has been shown to potentiate morphineanalgesia and possess some analgesic activity when used alone.

[0022] The invention includes compounds sharing the general formula:

Arg-Tyr-Tyr-Arg-Trp-Arg

[0023] (SEQ ID NO: 42) The compounds of the invention include compoundshaving substitutions to any one or two positions of the above formula.Specifically, the compounds of the invention include compounds havingnon-conservative substitutions and conservative substitutions, whereconservative substitutions involve the replacement of an amino acid byone with similar characteristics such that the substitution is unlikelyto substantially change the shape or properties of the peptide. Oneexample of conservative substitution is the substitution of onehydrophobic amino acid for another. Other substitutions arenon-conservative in nature. Still other substituted hexapeptidesaccording to the invention have non-natural, or modified amino acidssubstituted into the place of a natural or substituted amino acid.Finally, the compounds of the invention include hexapeptides withmodifications to the amino-terminus and/or carboxy-terminus of thehexapeptide.

[0024] One set of compounds of the invention includes compounds havingthe formula: Arg-Xaa-Tyr-Arg-Trp-Arg (SEQ ID NO: 17). In thesecompounds, “Xaa” is used to denote an amino acid substitution. Suitablesubstitutions include other natural amino acids, modified amino acids,and amino acid analogs. In one family of embodiments, the hexapeptidesinclude a modified Phe amino acid molecule in the Xaa position. In somespecific embodiments of the hexapeptide, Xaa is an amino acid selectedfrom the group consisting of Phe (4-Me) SEQ ID NO: 1, Phe (4-COOH) SEQID NO: 2, Phe (4-NO₂) SEQ ID NO: 3, Phe (4-F) SEQ ID NO: 4, Phe (4-CN)SEQ ID NO: 6.

[0025] In another family of hexapeptides according to the invention, Xaais a modified Tyr amino acid molecule. In specific embodiments, “Xaa”may be an amino acid molecule selected from the group consisting of Tyr(4-Me) SEQ ID NO: 5, Tyr (3-Cl) SEQ ID NO: 7, and Tyr (BN, 3-Cl) SEQ IDNO: 23.

[0026] Another set of compounds of the invention includes compoundshaving the formula: Arg-Tyr-Xaa-Arg-Trp-Arg (SEQ ID NO: 18). As above,“Xaa” denotes an amino acid substitution. Suitable substitutions includeother natural amino acids, modified amino acids, and amino acid analogs.In one family of embodiments, the hexapeptides include modified Pheamino acid molecules in the Xaa position. In some specific embodimentsof the hexapeptide, Xaa is an amino acid selected from the groupconsisting of Phe (4-F) SEQ ID NO: 12, Phe (NO₂) SEQ ID NO: 13, hPhe (2,4 di-NO₂) SEQ ID NO: 20, Phe (4-CH₂SO₃H) SEQ ID NO: 21, Phe (4-NHAc) SEQID NO: 16, and Phe (4-CH₂NH₂) SEQ ID NO: 22.

[0027] In another family of hexapeptides according to the invention, Xaais a modified Tyr amino acid molecule. In specific embodiments, Xaa maybe an amino acid molecule selected from the group consisting of Tyr (2,6 di-Me) (SEQ ID NO: 15).

[0028] The invention further includes yet another set of hexapeptides,these being according to the formula: Xaa₁-Tyr-Tyr-Xaa₂-Trp-Xaa₃ (SEQ IDNO: 19). In this family of hexapeptides, “Xaa₁”, “Xaa₂”, and “Xaa₃” areused to denote either the placement of Arg, or of an amino acidsubstitution. Hexapeptides within this group may have Arg at two of thethree positions and a substitution at the third. Alternatively,hexapeptides may have substitutions at two of the three positions andArg only at the remaining position. Finally, the hexapeptide may havesubstitutions at all three positions. As above, suitable substitutionsinclude other natural amino acids, modified amino acids, and amino acidanalogs. In some specific embodiments of the hexapeptide, Xaa₁, Xaa₂,and Xaa₃ are selected from the group consisting of Arg, ε-aminocaproyl,DAP, and DAB. In one such hexapeptide, Xaa₁ is ε-aminocaproyl, Xaa₂ isArg, and Xaa₃ is Arg (SEQ ID NO: 9). In another, Xaa₁ is Arg, Xaa₂ isε-aminocaproyl, and Xaa₃ is Arg (SEQ ID NO: 10). In still another, Xaa₁is Arg, Xaa₂ is Arg, and Xaa₃ is ε-aminocaproyl (SEQ ID NO: 11). Inanother, Xaa₁ is DAP, Xaa₂ is Arg, and Xaa₃ is Arg (SEQ ID NO: 14). Inyet another, Xaa₁ is DAB, Xaa₂ is Arg, and Xaa₃ is Arg (SEQ ID NO: 24).

[0029] Still another set of hexapeptide compounds according to theinvention include compounds having the formula: Arg-Tyr-Tyr-Arg-Xaa-Arg(SEQ ID NO: 27). In these compounds, “Xaa” is used to denote an aminoacid substitution. Suitable substitutions include other natural aminoacids, modified amino acids, and amino acid analogs. In one family ofhexapeptides of the invention, the Xaa is a modified Trp amino acidmolecule. In a specific embodiment of the invention, Xaa is Trp (5-CN)(SEQ ID NO: 8).

[0030] In addition to the amino acid substitutions outlined above, thehexapeptides of the invention may include amino-terminal and/orcarboxy-terminal modifications. Hexapeptides according to the inventionmay include modifications to both the amino and carboxy-termini, oralternatively may be made to only one terminus. In some embodiments, theamino terminus is modified to include a moiety selected from the groupconsisting of optionally substituted straight-chain alkyls, optionallysubstituted branched chain alkyls, aralalkyls, cycloalkyls, oralkylcycloalkyls containing from about 1 to about 12 carbon atoms. Insome preferred embodiments, the amino terminus of the peptide isacetylated.

[0031] The hexapeptides of the invention may additionally, oralternatively, be modified at the carboxy terminus. The carboxy-terminalmodifications may include an amine group, a carboxy group, a hydroxygroup, and aldehydes. Alternatively, the carboxy terminus of thehexapeptides may be modified to include substituted or non-substitutedstraight-chain alkyls, branched chain alkyls, aralalkyls, cycloalkyls,or alkylcycloalkyls containing from about 1 to about 12 carbon atoms.

[0032] The invention further includes pharmaceutical compositionscomprising one or more of the hexapeptides of the invention. Suchpharmaceutical compositions may additionally include a pharmaceuticallyacceptable diluent or excipient. Such a diluent or excipient may easedelivery and/or protect the hexapeptides from degradation during storageor administration.

[0033] The invention additionally includes methods of treating pain,including neuropathic pain. These methods comprise administering acompound comprising the hexapeptides of the invention to a patient inneed of analgesia.

[0034] These and other features and advantages of the present inventionwill become more fully apparent from the following description andappended claims, or may be learned by the practice of the invention asset forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order that the manner in which the above-recited and otherfeatures and advantages of the invention are obtained will be readilyunderstood, a more particular description of the invention brieflydescribed above will be rendered by reference to specific embodimentsthereof which are illustrated in the appended drawings. Understandingthat these drawings depict only typical embodiments of the invention andare not therefore to be considered to be limiting of its scope, theinvention will be described and explained with additional specificityand detail through the use of the accompanying drawings in which:

[0036]FIG. 1 is a chart showing the binding affinity of [³⁵S]GTPγSinduced by nociceptin, modified hexapeptides of the invention, andN¹-Phe-nociceptin (1-13)NH₂;

[0037]FIG. 2 is a chart showing the antagonist properties of IV-17-C(SEQ ID NO: 25), as shown by its ability to inhibit the stimulation of[³⁵S]GTPγS binding by nociceptin;

[0038]FIG. 3 shows the conformation of the agonist IV-16-C (SEQ ID NO:24) of the invention in comparison with a hypothesized nociceptinpharmacophore structure;

[0039]FIG. 4A shows the 3-dimensional hypothetical pharmacophore derivedfrom IV-16-C (SEQ ID NO: 24);

[0040]FIG. 4B shows the 3-dimensional hypothetical pharmacophore derivedfrom IV-17-C (SEQ ID NO: 25);

[0041] FIGS. 5A-5W compare the predicted structure of a group of peptideanalogs of the invention in comparison with the pharmacophore hypothesisfor IV-16-C (SEQ ID NO: 24);

[0042] FIGS. 6A-6W compare the predicted structure of a group of peptideanalogs of the invention in comparison with the pharmacophore hypothesisfor IV-17-C (SEQ ID NO: 25);

[0043] FIGS. 7A-7W show the 2-dimensional structures of a group ofpeptides of the invention;

[0044]FIG. 8 is a chart showing the agonist activity of VII-39-D and thepartial agonist activity of VII-43-C observed in a nociceptin-induced[³⁵S]GTPγS binding assay;

[0045]FIG. 9 is a chart showing the antagonist activity of VII-7-B ofright-shifting the dose/response curve for nociceptin stimulation of[³⁵S]GTPγS binding;

[0046]FIG. 10 is a chart showing the analgesic effects of the antagonistPentanoyl-RYYRWRNH₂ (SEQ ID NO: 26);

[0047]FIG. 11 is a set of charts showing that the Pentanoyl-RYYRWRNH₂(SEQ ID NO: 26) antagonist did not reverse the inhibition ofmorphine-induced analgesia brought about by nociceptin;

[0048]FIG. 12 is a chart showing the ability of the Pentanoyl-RYYRWRNH₂(SEQ ID NO: 26) antagonist to reduce morphine-induced analgesia at 5minutes post-injection;

[0049]FIG. 13 is a chart showing the ability of the Pentanoyl-RYYRWRNH₂(SEQ ID NO: 26) antagonist to reduce morphine-induced analgesia at 10minutes post-injection;

[0050]FIG. 14 is a chart showing the ability of the Pentanoyl-RYYRWRNH₂(SEQ ID NO: 26) antagonist to reduce morphine-induced analgesia at 20minutes post-injection;

[0051]FIG. 15 is a chart showing the pro-nociceptive effects of theadministration of the VII-87-B agonist (SEQ ID NO: 23);

[0052]FIG. 16 is a chart showing the dose-dependent reversal ofmorphine-induced analgesia by administration of the agonist VII-87-B(SEQ ID NO: 23);

[0053]FIG. 17 is a chart showing the effects of the administration ofthe agonist VII-39-D (SEQ ID NO: 7); and

[0054]FIG. 18 is a chart showing the attenuation of morphine-inducedanalgesia by agonist VII-39-D (SEQ ID NO: 7).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] The following detailed description of the embodiments of thehexapeptides of the present invention, as represented in FIGS. I through1 8, is not intended to limit the scope of the invention, as claimed,but is merely representative of presently preferred embodiments of theinvention.

[0056] The ORL-1 receptor and its natural ligand, nociceptin, provide anovel target for analgesic compounds. Animal studies have been conductedto confirm the usefulness of nociceptin agonists or antagonists withthis target in analgesic applications. These tests showed that agonistsor antagonists do exhibit analgesic properties. Specific testing showedeffectiveness of such compounds as an analgesic in models of neuropathicpain. Yamamoto et al., Neurosci. Lett., 224: 107-110 (1997).

[0057] Novel agonists and antagonists have been developed and aredisclosed herein. These compounds have been tested to more clearlycharacterize the effect of ORL1 activation and inhibition on analgesicand other opioid systems. These newly discovered antagonists may laterbe used to determine whether nociceptin has constitutive activity inmammalian brain, as well as whether an ORL1 receptor antagonist will actas a non-addicting analgesic. Further, the compounds of the inventionmay exhibit diuretic properties, as well as cancer-fighting ability.

[0058] As noted above, the identification and characterization ofhigh-affinity compounds furthers the development of a betterunderstanding of the actions of nociceptin and its receptor ORL1. Thephysiological actions of nociceptin are poorly understood in partbecause of the absence of low molecular weight, stable agonists andhigh-affinity antagonists. Although initial testing showed thatnociceptin decreased tail flick latencies in rats, and further that itinhibited opiate analgesia, further testing has begun to show that thephysiological actions of nociceptin may be very complicated. Somestudies showed the anti-opiate activity of nociceptin to potentially beregion- and assay-specific. In one study, nociceptin was found to beanalgesic in the spinal cord. Tian et al., Br. J. Pharmacol.,120:676-680(1997); Xu et al, Neuroreport, 7:2092-2094 (1996).Additionally, as noted above, it has also been reported to be aneffective analgesic in a model of chronic pain. Yamamoto et al.,Neurosci. Lett., 224: 107-110 (1997). In addition, nociceptin exhibitssignificantly greater potency as an analgesic when used in diabetic micethan when used in non-diabetic mice. Kamei, et al., Eur. J. Pharmacol.,370:109-116 (1999). In contrast, it has also been shown to induceallodynia, a condition in which normal non-painful stimuli cause pain,when injected into the spinal cord. Hara et al., Br. J. Pharmacol.,121:401-408 (1997).

[0059] For research purposes, the development of antagonists will beeven more valuable. As with opiate receptors, and now withtetrahydrocannabinol (THC) receptors, the availability of an antagonistallows for a better understanding of the specific actions of a compound.Opiate actions are currently defined by their ability to be antagonizedby naloxone. It is anticipated that the same criteria apply for ORL1.The availability of an antagonist also aids in the identification of anyconstitutive actions of nociceptin-ORL1, or in vivo actions brought onby altered physiologic states. For instance, naloxone has no effects onanalgesia in naive animals, but it does have important effects withrespect to cerebral glucose utilization in specific brain regions. Krauset al., Brain Res., 724:33-40 (1996). These experiments demonstrateregions of endogenous opiate activity in untreated animals. Of course,naloxone has significant and well known effects in animals with alteredphysiologic states, including the precipitation of withdrawal and theinhibition of stress-induced analgesia. Antagonists to ORL1 willundoubtedly uncover many actions of nociceptin in normal and alteredstates.

[0060] The invention thus provides hexapeptide compounds which interactwith the ORL-1 receptor (hereinafter, the “nociceptin receptor”,including compounds exhibiting agonist and antagonist properties. Thehexapeptides of the invention may be constructed solely of natural aminoacids. Alternatively, the hexapeptides may include non-natural aminoacids including, but not limited to, modified amino acids. Modifiedamino acids include natural amino acids which have been chemicallymodified to include a group or groups not naturally present on the aminoacid. The hexapeptides of the invention may additionally include D-aminoacids. Still further, the hexapeptides of the invention may includeamino acid analogs.

[0061] A first group of these compounds were constructed having thegeneral formula:

Arg-Tyr-Tyr-Arg-Trp-Arg

[0062] These compounds contained various amino- and carboxy-terminalmodifications and an amino acid substitution, as shown in Table 1. TABLE1 Binding Affinity and Functional Activity of Compounds at ORL1.[³H]nociceptin SEQ Binding [³⁵S]GTPγS Binding ID IC₅₀ EC₅₀ PercentCompound NO: (nM) (nM) Stimulation Nociceptin 1.0 4.2 100 Ac-RYYRWR-NH₂43 0.72 1.2 100 IV-12-B Ac-RY(D)YR(D)WNH₂ 44 1145 >10,000 <10 IV-15-AButryl-RYYRWR-NH₂ 45 4.3 35.3 63 IV-16-C Propionyl-RYYRWR-NH₂ 24 1.222.3 82 IV-17-C Hexanoyl-RYYRWR-NH₂ 25 2.6 * 18 IV-18-CHeptanoyl-RYYRWR-NH₂ 26 2.6 16.9 54

[0063] The compounds of Table 1 were tested for [³⁵S]GTPγS stimulationsimilar to that induced by nociceptin. The results of this testing areshown in the chart in FIG. 1. Nociceptin stimulates [³⁵S]GTPγS to bindto membranes derived from CHO cells transfected with human ORL-1. Thecompounds of Table 1 were tested in a [³⁵S]GTPγS binding assay todetermine their ability to stimulate [³⁵S]GTPγS binding in comparison tonociceptin.

[0064] The [³⁵S]GTPγS binding assays were conducted generally asdescribed by Traynor and Nahorski (1995). First, CHO cells transfectedwith human ORL-1 are scraped from tissue culture dishes into 20 mMHEPES, 1 mM EDTA, and then centrifuged at 500×g for 10 minutes. Thecells are then re-suspended in this buffer and homogenized using aPolytron Homogenizer.

[0065] The cellular homogenate is next centrifuged at 20,000×g for 20minutes. Following this, the resulting pellet is resuspended in a buffercontaining 20 mM HEPES, 10 mM MgCl₂, and 100 mM NaCl, having a resultingpH of 7.4. The suspension is then re-centrifuged at 20,000×g andsuspended once more in the above-listed buffer. The pellet may be frozenat −70° C. prior to the final centrifugation. For the binding assay,membranes (10-20 μg protein) are incubated with [³⁵S]GTPγγS (50 pM), GDP(usually 10 μM), and the desired compound. The total volume of themixture is 1 ml, which is incubated for 60 min at 25° C.

[0066] Following incubation, samples are filtered over glass fiberFilters and counted. A dose response with the full agonist nociceptinmay then be conducted in each experiment to identify full and partialagonist compounds.

[0067] As seen in FIG. 1 and Table 1, IV-16-C (SEQ ID NO: 24), havingthe structure Propionyl-RYYRWR-NH₂ exhibited high affinity and appearedto be a potent, nearly full agonist of ORL-1. In contrast, compoundIV-17-C (SEQ ID NO: 25), having the structure Hexanoyl-RYYRWR-NH₂maintains high affinity but is a very low efficacy compound.

[0068] As seen in FIG. 1, the antagonist properties of IV-17-C (SEQ IDNO: 25) can readily be observed in its ability to inhibit thestimulation of [³⁵S]GTPγS binding induced by 10 nM nociceptin. The 20%stimulation found at 1 and 10 μM of [³⁵S]GTPγS confirms the partialagonist activity of this compound, as also seen in FIG. 1 and shown inTable 1.

[0069] In addition, as seen in FIG. 2, IV-17-C (SEQ ID NO: 25) is atleast 10 times more potent as an antagonist than the complete antagonistN′-Phe-nociceptin (1-13)NH₂ reported in Calo et al., 2000.N¹-Phe-nociceptin (1-13)NH₂ is a recently-developed peptide antagonistthat has been shown to potentiate morphine analgesia and to possess someanalgesic activity on its own. IV-17-C (SEQ ID NO: 25) was tested invivo for analgesic activity and for potentiation of morphine analgesia.This testing showed no measurable in vivo activity. Without beinglimited to any one theory, it was concluded that the apparent in vivoinactivity of the molecule is likely attributable to its rapid in vivodegradation. These studies suggest that IV-17-C (SEQ ID NO: 25) is avery promising lead, for which more stable analogs have potential asanalgesic compounds.

[0070] Based upon the activity of the compounds discussed above,additional compounds were synthesized. These compounds were varied inorder to identify residues which must be conserved in order to retainbinding affinity and functional activity. Thus, the group consisted ofmolecules resulting from “alanine scans” of the high affinity agonistIV-16-C (SEQ ID NO: 24) and the antagonist IV-17-C (SEQ ID NO: 25). Inthese alanine scans, the original sequences, Propionyl-RYYRWR-NH₂ (SEQID NO: 24) and Hexanoyl-RYYRWR-NH₂ (SEQ ID NO: 25) were systematicallymodified by substituting an alanine amino acid into every position ofthe hexapeptide, one amino acid at a time. The binding affinities ofthese alanine scan molecules are shown in Table 2. TABLE 2 BindingAffinitics of Alanine Scan of IV-16-C and 1V-17-C. SEQ ID Compound NO:IC₅₀ (nM) IV-21-C Propionyl-AYYRWR-NH₂ 46 1780 IV-23-BPropionyl-RAYRWR-NH₂ 47 182 IV-25-B Propionyl-RYARWR-NH₂ 48 495 IV-27-BPropionyl-RYYAWR-NH₂ 49 400 IV-29-B Propionyl-RYYRAR-NH₂ 50 1890 IV-31-BPropionyl-RYYRWA-NH₂ 51 76 IV-33-B Hexanoyl-AYYRWR-NH₂ 52 1015 IV-35-BHexanoyl-RAYRWR-NH₂ 53 113 IV-37-B Hexanoyl-RYARWR-NH₂ 54 79 IV-39-BHexanoyl-RYYAWR-NH₂ 55 1000 IV-41-B Hexanoyl-RYYRAR-NH₂ 56 710 IV-43-BHexanoyl-RYYRWA-NH₂ 57 311 Nociceptin 1.3

[0071] The data in Table 2 indicate the importance of each residue inthe parent peptide, Ac-RYYRWR-NH₂, even with the lipophilic addition tothe amino terminals. Even in the best cases, binding affinities droppedby a factor of at least 30.

[0072] The IV-16-C (SEQ ID NO: 24) agonist was then subjected tocomputational studies. First the hexapeptide molecule was modeled in arandom conformation using software model building and energy refinementtools. The software utilized was CATALYST, from Molecular Simulations,Inc. These structural models were used to create an arbitrary3-dimensional pharmacophore model. This was done using the functionalmapping capability of the molecular modeling program used in the “viewhypothesis workbench” mode of CATALYST. FIG. 3 shows the 3-dimensionalconformation of the IV-16-C peptide overlapped with the 3-dimensionalstructure of the hypothetical agonist pharmacophore.

[0073] These preliminary computational studies were conducted with nodata available about the structure of the peptide that had been deducedfrom experimental sources (NMR, X-ray). As a result, a completely randomconformation was chosen for the purpose of illustration in FIG. 3.

[0074] The IV-16-C (SEQ ID NO: 24) peptide studied above is a veryflexible molecule and in principle may likely adopt many low energyconformations, due to the fact that it is endowed with at least 15rotatable bonds. Pharmacophore generation methods in CATALYST aresensitive to the conformational models employed. Hence, the choice ofthe pharmacophore for the purpose of illustrations of database searchmethods is purely random. The actual pharmacophore structure may bederived based on an experimental structure for such flexible molecules.

[0075] The pharmacophore may be deduced by conducting a conformationalsearch to find molecules similar to leading compounds such as IV-16-C(SEQ ID NO: 24) and creating a multi-conformation 3-dimensional databaseof the molecules. The compound may then be subjected to an alignment tofit, and the quality of the molecules categorized be assessed withrespect to the lead compounds. These data help to generate apharmacophore model, which may then be studied using databases such asthe Available Chemical Directory (ACD), BioByte Master File, NationalCancer Institute Database (NCI), The Derwent World Drug Index, and theMaybridge catalog. Accuracy may then be assessed by producing orlocating molecules conforming to the pharmacophore model, mappingbetween the pharmacophore model and the new molecules, predicting theactivity of the new molecules based on the pharmacophore, andsynthesizing and assaying the more promising drug candidates.

[0076] Following the above plan, a series of peptide analogs wasgenerated for testing. These analogs incorporated amino acidreplacements using commercially-available non-natural amino acids. Thesequences of these analogs are listed in Table 3. TABLE 3 Sequences ofthe Peptides Synthesized in Year 02 SEQ Peptide ID No. NO: SequenceVII-1-A  28 Nipacotyl-Arg-Tyr-Tyr-Arg-Trp-Arg-NH₂ VII-2-A  29β-Nva-Arg-Tyr-Tyr-Arg-Trp-Arg-NH₂ VII-3-A  30β-aminoisobutryl-Arg-Tyr-Tyr-Arg-Trp-Arg-NH₂ VII-4-B  311-aminocyclohexanoyl-Arg-Tyr-Tyr-Arg-Trp-Arg-NH₂ VII-7-B  26Pentanoyl-Arg-Tyr-Tyr-Arg-Trp-Arg-NH₂ VII-11-B 32Ac-Arg-Phg-Phg-Arg-Trp-Arg-NH₂ VII-13-B 33Ac-Arg-Tyr-Phg-Arg-Trp-Arg-NH₂ VII-15-B 1Ac-Arg-Phe(4-Me)-Tyr-Arg-Trp-Arg-NH₂ VII-17-B 34Ac-Arg-Phe(4-Me)-Phe(4-Me)-Arg-Trp-Arg-NH₂ VII-19-B 2Ac-Arg-Phe(4-COOH)-Tyr-Arg-Trp-Arg-NH₂ VII-21-C 35Ac-Arg-Phe(4-COOH)-Phe(COOH)-Arg-Trp-Arg-NH₂ VII-23-B 3Ac-Arg-Phe(NO₂)-Tyr-Arg-Trp-Arg-NH₂ VII-27-B 36Ac-Arg-Phe(SO₃H)-Tyr-Arg-Trp-Arg-NH₂ VII-29-B 37Ac-Arg-Phe(4-SO₃H)-Tyr-Arg-Trp-Arg-NH₂ VII-31-B 4Ac-Arg-Phe(4-F)-Tyr-Arg-Trp-Arg-NH₂ VII-33-B 38Ac-Arg-Phe(4-F)-Phe(4-F)-Arg-Trp-Arg-NH₂ VII-35-C 5Ac-Arg-Tyr(4-Me)-Tyr-Arg-Trp-Arg-NH₂ VII-37-B 39Ac-Arg-Tyr(4-Me)-Tyr(4-Me)-Arg-Trp-Arg-NH₂ VII-39-D 7 Ac-Arg-Tyr(BN*,3-Cl)-Tyr-Arg-Trp-Arg-NH₂ VII-43-C 6Ac-Arg-Phe(4-CN)-Tyr-Arg-Trp-Arg-NH₂ VII-49-B 40Ac-Arg-hPhe-hPhe-Arg-Trp-Arg-NH₂ VII-51-A 8Ac-Arg-Tyr-Tyr-Arg-Trp(5-CN)-Arg-NH₂ VII-53-B 9Ac-ε-aminocaproyl-Tyr-Tyr-Arg-Trp-Arg-NH₂ VII-55-A 10Ac-Arg-Tyr-Tyr-ε-aminocaproyl-Trp-Arg-NH₂ VII-57-C 11Ac-Arg-Tyr-Tyr-Arg-Trp-ε-aminocaproyl-NH₂ VII-61-B 12Ac-Arg-Tyr-Phe(4-F)-Arg-Trp-Arg-NH₂ VII-63-B 13Ac-Arg-Tyr-Phe(4-NO₂)-Arg-Trp-Arg-NH₂ VII-65-F 14Ac-Dap-Tyr-Tyr-Arg-Trp-Arg-NH₂ VII-67-A 41Ac-Dab-Tyr-Tyr-Arg-Trp-Arg-NH₂ VII-71-B 20Ac-Arg-Tyr-hPhe(2,4-di-NO₂)-Arg-Trp-Arg-NH₂ VII-73-A 15Ac-Arg-Tyr-Tyr(2,6-di-Me)-Arg-Trp-Arg-NH₂ VII-75-B 21Ac-Arg-Tyr-Phe(4-CH₂SO₃H)-Arg-Trp-Arg-NH₂ VII-77-A 16Ac-Arg-Tyr-Phe(4-NHAc)-Arg-Trp-Arg-NH₂ VII-79-A 22Ac-Arg-Tyr-Phe(4-CH₂NH₂)-Arg-Trp-Arg-NH₂ VII-87-B 23Ac-Arg-Tyr(3-Cl)-Tyr-Arg-Trp-Arg-NH₂

[0077] All of the peptides listed above in Table 3 were next synthesizedusing Merrifield's Solid Phase technique on a CS Bio 136 PeptideSynthesizer. Fmoc-Rink-Amide resin was purchased from AnaSpec (San Jose,Calif.). Fmoc amino acids were purchased from AnaSpec or PerSeptiveBiosystems (Foster City, Calif.). The non-natural or unusual amino acidsneeded were purchased from RSP Amino Acids Analogues Inc. The purity ofpeptides was checked by analytical high pressure liquid chromatography(HPLC) and Mass Spectroscopy and they were greater than 95% pure.

[0078] These molecules were subjected to computational analysis topredict their potential utility. The molecules were designed and modeledin CATALYST in “view compound workbench” mode. As a reference, the3-dimensional structure of the compounds IV-16-C (SEQ ID NO: 24) andIV-17-C (SEQ ID NO: 25) are shown overlapped with their predicted3-dimensional pharmacophores in FIGS. 4A and 4B, respectively. Thestructures of the newly-generated analogs were compared with these twopharmacophores, using the “compare fit” function of CATALYST. FIGS. 5Athrough 5W show the results of overlapping the predicted 3-dimensionalstructures of the hexapeptide analogs with the predicted pharmacophoreof IV-16-C (SEQ ID NO: 24). Similarly, FIGS. 6A through 6W show theresults of overlapping the predicted 3-dimensional structures of thehexapeptide analogs with the predicted pharmacophore of IV-17-C (SEQ IDNO: 25). FIGS. 7A through 7W show the simple 2-dimensional structures ofeach of the hexapeptide analog compounds investigated.

[0079] The results of the computational studies are shown in Table 4. Inthis table, the structures of the analog hexapeptides were compared withthe pharmacophores using the “compare fit” function of CATALYST. Usingthese methods, best fit values ranging from 2.26 to 5.99 were obtained,the higher values indicating a better overlap of the pharmacophore“Hypothesis” and the analog and the lower values indicating a worseoverlap. For example, value of zero indicates no overlap while a valueof six indicates a perfect overlap. TABLE 4 Results of ComputationalExperiments on Peptide Analogs Conformational Best Fit Best Fit CompoundEnergy IV-16-C 1V-17-C Name (KCal/mol) Hypothesis Hypothesis VII-1-A81.23 4.91 5.99 VII-2-A 78.87 4.82 5.99 VII-3-A 82.51 3.82 3.95 VII-4-B81.22 4.66 5.98 VII-7-B 78.17 4.92 5.97 VII-9-A 86.01 3.98 3.97 VII-11-B85.13 3 2.75 VII-13-B 78.32 3.96 2.99 VII-15-B 145.90 3.25 3.79 VII-17-B81.00 2.26 2.92 VII-19-B 204.71 3.82 3.96 VII-21-C 207.12 2.97 2.98VII-23-B 205.06 3.77 3.97 VII-25 150.5 2.99 2.98 VII-27-B 144.71 3.733.92 VII-29-B 146.01 2.98 2.99 VII-31-B 145.33 3.27 3.94 VII-33-B 145.023.00 3.00 VII-35-C 81.22 3.98 5 VII-37-B 81.28 3.00 3.99 VII-39-D 95.103.98 4.98 VII-41 107.13 2.99 3.99 VII-43-C 75.53 3.18 2.99

[0080] Receptor binding studies were conducted on human ORL1 (opiatereceptor like 1) transfected into Chinese hamster ovary (CHO) cellsusing each of the hexapeptide analog compounds of Table 3. All thecompounds were evaluated for binding affinities. The results are shownin Table 5. Affinity was determined using [³H] nociceptin binding tomembranes derived from CHO cells transfected with human ORL-1. IC₅₀values and Hill coefficients were then determined using the curvefitting program Prism, and Ki values were calculated from the formulaKi=IC₅₀/(1+L/Kd) (Chang and Prusoff), where Kd is the binding affinityof [³H]nociceptin and L is the concentration of [³H]nociceptin in eachparticular experiment. [L] of nociceptin was approximately 0.2 nM, andthe Kd, as determined by the Scatchard analysis is 0.05 nM. The datashown in Table 5 represents the average±SEM of at least two experimentsconducted in triplicate.

[0081] ORL1-containing CHO cells were produced using cDNA obtained fromDr. Brigitte Kieffer. The cells are grown in Dulbecco's Modified EagleMedium (DMEM) with 10% fetal bovine serum, in the presence of 0.4 mg/mlG418 and 0.1% penicillin/streptomycin, in 100-mm plastic culture dishes.For binding assays, the cells are scraped off the plate at confluence.For determination of inhibition of cAMP accumulation, cells aresubcultured onto 24-well plates and used at confluence.

[0082] Receptor binding assays will be examined as described previouslyin Toll, 1992. Cells are removed from the plates by scraping with arubber policeman, and then homogenized in Tris buffer using a Polytronhomogenizer. Following this homogenization step, the cellular mixture iscentrifuged once and washed by an additional centrifugation at 40,000×gfor 15 min. The pellet formed during the centrifugation is re-suspendedin 50 mM Tris, pH 7.5. The resulting suspension is incubated with[³H]nociceptin in a total volume of 1.0 ml, in a 96-well format, for 120min at 25° C. Samples of the suspension are then filtered over glassfiber filters using a Wallac cell harvester.

[0083] For the ORL-1 binding experiments, 1 mg/ml bovine serum albuminis used to prevent absorption of the ligand to the glass tubes, andfilters are soaked in 0.1% polyethyleneimine (PEI) to prevent adsorptionto the glass fiber filters, thus lowering nonspecific bindingconsiderably. TABLE 5 Binding Affinities of Novel Peptide Analogs atORL1 Compound K₁ (nM) ± SEM Hill Coefficient Nociceptin 0.04 ± 0.005 1.0VII-1-A  30.3 ± 2.9 0.97 VII-2-A  11.8 ± 1.9 1.0 VII-3-A  1.18 ± 0.180.72 VII-4-B  9.25 ± 2.05 1.0 VII-7-B  0.16 ± 0.05 0.8 VII-11-B 30.4 ±3.62 0.93 VII-13-B 27.0 ± 3.85 0.73 VII-15-B 0.48 ± 0.18 0.79 VII-17-B10.05 ± 1.44 1.06 VII-19-B 40.6 ± 15.9 1.0 VII-21-C >10,000 VII-23-B0.44 ± 0.26 0.74 VII-27-B 155 ± 15.2 1.12 VII-29-B >10,000 VII-31-B 0.52± 0.13 0.81 VII-33-C 1.51 ± 0.005 0.89 VII-35-C 0.43 ± 0.05 0.75VII-37-B 6.29 ± 0.15 0.82 VII-39-D 0.03 ± 0.02 1.03 VII-43-C 0.15 ± 0.020.73 VII-49-B 5.49 ± 0.0073 0.94 VII-51-A 0.3 ± 0.1333 0.7 VII-53-B29.09 ± 0.0014 0.86 VII-55-A 39 ± 0.001 1.12 VII-57-C 41.93 ± 0.001 0.95VII-61-B 0.05 ± 0.8 1.05 VII-63-B 0.17 ± 0.2353 1.01 VII-65-F 28.1 ±0.0014 1.1 VII-67-A 5.59 ± 0.0072 1.07 VII-71-B 0.63 ± 0.0635 0.88VII-73-A 0.04 ± 1 0.95 VII-75-B 26.69 ± 0.0015 0.75 VII-77-A 0.88 ±0.0455 0.62 VII-79-A 2.54 ± 0.0157 0.89 VII-87-B 0.26±

[0084] For the data in Table 5, binding was conducted as describedabove. IC₅₀ values and Hill coefficients were determined using thecurve-fitting program Prism. K₁ values were calculated using theequation K₁=IC₅₀/(1+[L]/Kd). [L] of nociceptin was approximately 0.2 nM,and the K_(d), as determined by Scatchard analysis, was 0.05 nM. Thedata in Table 5 represent the average±SEM of at least two experimentsconducted in triplicate.

[0085] Table 6 shows the results of [³⁵S]GTPγS binding assays conductedusing the compounds of Table 3. [³⁵S]GTPγS binding is conductedgenerally according to the methods described by Traynor and Nahorski(1995). First, cells are scraped from their tissue culture dishes into20 mM HEPES, 1 mM EDTA. This suspension is then centrifuged at 500×g for10 minutes. Following this, the cells were re-suspended in buffer andhomogenized using a Polytron Homogenizer. The resulting homogenate wascentrifuged at 20,000×g for 20 minutes. The pellet produced duringcentrifugation is next re-suspended in a buffer containing 20 mM HEPES,10 mM MgCl₂, and 100 mM NaCl, having a pH of 7.4. The suspension isre-centrifuged at 20,000×g and then suspended once more in the bufferoutlined above. The pellet may be frozen at −70° C. prior to the finalcentrifugation. For the binding assay, membranes (10-20 μg protein) areincubated with [⁵S]GTPγS (50 pM), GDP (usually 10 μM), and the desiredcompound, in a total volume of 1 ml, for 60 minutes at 25° C. Samplesare filtered over glass fiber filters and counted as described for thebinding assays. A dose response with the full agonist nociceptin wasthen conducted in each experiment to identify full and partial agonistcompounds. TABLE 6 Stimulation of |^(±)S|GTPγS Binding of PeptideAnalogs in CHO Cell Membranes Transfected with ORLi Compound EC50 (nM) ±SEM % Stimulation ± SEM Nociceptin 0.5 ± 0.001 100 VII-1-A  FLAT <20VII-2-A  FLAT <20 VII-3-A  68 ± 0 29.1 ± 1.9 VII-4-B  FLAT <20 VII-7-B FLAT <20 VII-15-B 18.8 ± 0.5 59.8 ± 16.2 VII-17-B FLAT <20 VII-19-B 854± 29.7 45.5 ± 8 VII-23-B 15.7 ± 0.6‘ 56.6 ± 20.4 VII-31-B 35.7 ± 0.254.4 ± 1.2 VII-33-C FLAT <20 VII-35-C 51.1 ± 11.6 52.1 ± 0.6 VII-37-BFLAT <20 VII-39-D 0.3 ± 0.08 89 ± 7.1 VII-43-C 29.5 ± 8.0 49.7 ± 6.6VII-49-B FLAT <20 VII-51-A 156.1 ± 64.39 49.1 ± 0.19 VII-53-B 167.3 ±103.73 39.5 ± 4 VII-55-A 1230.8 ± 1007.25 26.6 ± 1.95 VII-57-C 358 ±5.15 37.4 ± 2.64 VII-61-B 2 ± 0.26 70.4 ± 1.98 VII-63-B 3.2 ± 0.65 43.2± 0.59 VII-65-F FLAT 13.7 ± 1.175 VII-67-A FLAT <20 VII-71-B 7.5 ± 0.033.65 ± 16.83 VII-73-A 5.2 ± 0.24 52.7 ± 0.175 VII-75-B FLAT <20VII-77-A 115.1 ± 55.055 19 ± 6.92 VII-79-A FLAT <20 VII-87-B 3.9 72

[0086] In these [³⁵S]GTPγS assays, binding was conducted as describedabove. EC₅₀ values and percent stimulation were determined using theprogram Prism. The data shown represent the average±SEM of at least twoexperiments conducted in triplicate. If percent stimulation was lessthan 20%, EC₅₀ values could not be reliably determined, and the compoundwas considered an antagonist.

[0087] As seen in Table 5, the structural modifications made in peptideanalogs have produced a variety of receptor affinities, potencies, andefficacies. The highest affinity compound (VII-39-D) (SEQ ID NO: 7) hasa Ki value of 0.03 nM, equivalent to that of nociceptin. Themodifications also produced compounds ranging from a full agonist(VII-39-D) (SEQ ID NO: 7), to several antagonists. The activity ofseveral of these compounds is compared in FIG. 8.

[0088] Specifically, FIG. 8 shows stimulation of [³⁵S]GTPγS binding bythe full agonist VII-39-D (SEQ ID NO: 7), the partial agonist VII-43-C(SEQ ID NO: 6), and the full agonist, standard nociceptin. As can beseen in Table 5 and FIG. 8, VII-39-D (SEQ ID NO: 7) also has potencysimilar to that of nociceptin. The most potent antagonist VII-7-B (SEQID NO: 26) has been tested for antagonist potency by Schild analysis. Asseen in FIG. 9, VII-7-B (SEQ ID NO: 26) produces a dose-dependentparallel shift in the nociceptin dose response curve. This indicatescompetitive inhibition. Schild analysis produced the following values:Ke=1.06±0.11, slope=−1.02 (competitive inhibition), pA₂=8.99±0.05. Thiscompound is more potent as an antagonist when tested in vitro than anyantagonist found in the literature to date.

[0089] As briefly discussed above, peptide analog VII-39-D (SEQ ID NO:7) is a very potent agonist. An additional hexapeptide analog VII-87-B(SEQ ID NO: 23) was similarly tested and shown to be an agonist.Agonists have been shown to have efficacy as anxiolytics against someforms of chronic pain when administered intrathecally. VII-7-B (SEQ IDNO: 26) is a very potent antagonist. Some such antagonists have beenshown to be effective in animal thermal pain models, particularly whenadministered into the brain.

EXAMPLES

[0090] The above hexapeptide drugs were used in in vivo experimentationto show their potential medical usefulness. Specifically, the antagonistVII-7-B (SEQ ID NO: 26), the agonist 87-B (SEQ ID NO: 23), and theagonist VII-39-D (SEQ ID NO: 7) were tested in vivo alone or incombination with morphine. The antagonist VII-7-B (SEQ ID NO: 26) wasalso tested in combination with morphine and N/OFQ.

[0091] Nociception was assessed using a tail flick assay with mice kepton a 12-hours light and 12-hours dark regimen and housed 10 per cage.Tail flick latencies were determined using a Tail Flick AnalgesiaInstrument (Stoelting) that uses radiant heat. This instrument isequipped with an automatic quantification of tail flick latency and a15-second cutoff to prevent damage to the animal's tail. During testing,the focused beam of light was applied to the lower half of the animal'stail, and tail flick latency was recorded. Baseline values for tailflick latency were determined before drug administration in each animal.Basal tail flick latency was between 3.7 and 6.3 seconds (average4.6±0.1 SEM). Immediately after testing, animals were lightlyanaesthetized with isoflurane and received a unilateral 2 μlintracerebroventricular injection approximately 2.0 mm caudal andapproximately 2.0 mm lateral with respect to the bregma (the junction ofthe sagittal and coronal sutures of the skull), and 3 mm ventral fromthe skull surface). Injections may be made using a Hamilton syringeequipped with a 26-guage needle fitted with a plastic sleeve to preventmore than 2.5 mm penetration beyond the skull surface. Following theintracerebroventricular injections, the animals were tested for tailflick latencies at 5-, 10-, and 20-minutes post-injection.

[0092] Antinociception was quantified by the following formula:

% Antinociception=100*[(test latency-baseline latency)/(15−baselinelatency)].

[0093] If the animal subject did not respond prior to the 15-secondcutoff, the animal was assigned a score of 100%.

[0094] Behavioral results were analyzed using ANOVAs with theantagonist, agonist, morphine, and N/OFQ as between group variables andpost-drug treatment time (5-, 10-, and 20-minutes) as the repeatedmeasure followed by Dunnet post-hoc tests where appropriate. The levelof significance was set at p<0.05.

[0095] In the experiments examining the combined effects of morphinealone or with the antagonist and/or N/OFQ, planned comparisons were usedto compare the effects of combined administration of antagonist/ N/OFQand morphine to the morphine alone groups at the three differentpost-infusion time points since it was hypothesized that the antagonistand/or N/OFQ would alter morphine-induced analgesia. Also, plannedcomparisons were used to compare the groups that received N/OFQ andmorphine since it was hypothesized that the antagonist would decreasethe efficiency of N/OFQ on morphine-induced analgesia. The modifiedBoniferroni Test was used for these planned comparisons (p value was setat P<0.036). Doses were determined based on the potency of the compoundstested.

Example 1

[0096] In a first Example, the antagonist VII-7-B (SEQ ID NO: 26) havingthe sequence: Pentanoyl-RYYRWR-NH₂ was assayed for analgesic effects. Inthis assay, the responses of a control mouse were compared against micereceiving three different dosages of the VII-7-B antagonist (SEQ ID NO:26). The responses were measured at 5, 10, and 20 minutes after theintracerebroventricular injection of antagonist. The test and baselinelatencies were then used to calculate the antinociception as detailedabove. In the figure, an asterisk represents a significant difference ofa test animal from the respective controls. Here, the antagonist VII-7-B(SEQ ID NO: 26) showed analgesic properties in those mice receiving the10.0 and 30.0 nmol intracerebroventricular injections at 10 and 20minutes post-injection.

Example 2

[0097] The antagonist was next assayed for the ability to reverse theinhibition of morphine-induced analgesia. In this assay, the controlreceived morphine alone, while test animals received morphine+3 nmol ofthe antagonist, morphine+10 nmol of the antagonist. These results werecompared with test animals receiving morphine+3 nmol nociceptin,morphine+nociceptin+3 nmol antagonist, and morphine+nociceptin+10 nmolantagonist. For each of these animals, response was measured at 5, 10,and 20 minutes after intracerebroventricular injection.

[0098] As shown in FIG. 11, the antagonist did not appear tosignificantly reverse the inhibition of morphine-induced analgesia.Little reversal was observed in the two animals receiving morphine andantagonist. Some reversal appears to be present in the animals receivingmorphine, antagonist, and nociceptin. This result was explored further.

[0099] Referring now to FIG. 12, at 5 minutes post-injection, theadministration of 10 nmol of the VII-7-B (SEQ ID NO: 26) antagonistalone, as well as in combination with N/OFQ resulted in a reduction inmorphine-induced analgesia. In this Figure, as above, asterisksrepresent a significant difference from morphine alone. As seen in FIGS.13 and 14, however, at 10 and 20 minutes post injection, the antagonistdid not alter the effects of nociceptin.

Example 3

[0100] The agonist 87-B was next assayed for analgesic effects. In thisassay, the responses of a control mouse were compared against micereceiving three different dosages of the VII-87-B agonist (SEQ ID NO:23). The responses were measured at 5, 10, and 20 minutes after theintracerebroventricular injection of agonist. The test and baselinelatencies were then used to calculate the antinociception as detailedabove. The results of this assay are shown in FIG. 15. In the figure, anasterisk represents a significant difference of a test animal from therespective controls.

[0101] Here, the agonist VII-87-B (SEQ ID NO: 23) inducedpro-nociception in mice receiving 10 nmol of agonist byintracerebroventricular injection at 5, 10, and 20 minutespost-injection.

[0102] The agonist VII-87-B was further investigated by evaluating itsability to reverse morphine-induced analgesia. In this assay, thecontrol animal received 10 nmols of morphine alone, while test animalsreceived morphine+0.1 nmol of the agonist, morphine+1.0 nmol of theagonist, or morphine+10.0 nmol of the agonist. For each of theseanimals, response was measured at 5, 10, and 20 minutes afterintracerebroventricular injection.

[0103] The results of this assay are shown in FIG. 16. This assay showeddose-dependent reversal of morphine-induced analgesia at 10 and 20minutes by the antagonist in animals injected with 10 nmol of antagonistin addition to the morphine.

Example 4

[0104] The agonist 39-D was next assayed for analgesic effects. In thisassay, the responses of a control mouse were compared against micereceiving three different dosages of the VII-39-D agonist (SEQ ID NO:7). The responses were measured at 5, 10, and 20 minutes after theintracerebroventricular injection of agonist. The test and baselinelatencies were then used to calculate the antinociception as detailedabove. The results of this assay are shown in FIG. 17.

[0105] The agonist VII-39-D (SEQ ID NO: 7) did not induce anti- orpro-nociception in mice receiving 0.1, 1.0, or 10.0 nmol of agonist byintracerebroventricular injection at 5, 10, and 20 minutespost-injection.

[0106] The agonist VII-39-D was then further investigated by evaluatingits ability to reverse morphine-induced analgesia. In this assay, thecontrol animal received 10 nmols of morphine alone, while test animalsreceived morphine+0.1 nmol of the agonist, morphine+1.0 nmol of theagonist, or morphine+10.0 nmol of the agonist. For each of theseanimals, response was measured at 5, 10, and 20 minutes afterintracerebroventricular injection. The results of this assay are shownin FIG. 18. This assay showed attenuation of morphine-induced analgesiaat 5, 10, and 20 minutes by the agonist in animals injected with 1.0 and10 nmol of agonist in addition to the morphine.

[0107] The present invention may be embodied in other specific formswithout departing from its structures, methods, or other essentialcharacteristics as broadly described herein and claimed hereinafter. Thedescribed embodiments are to be considered in all respects only asillustrative, and not restrictive. The scope of the invention is,therefore, indicated by the appended claims, rather than by theforegoing description. All changes that come within the meaning andrange of equivalency of the claims are to be embraced within theirscope.

1 57 1 6 PRT Artificial VII-15-B Hexapeptide 1 Arg Xaa Tyr Arg Trp Arg 15 2 6 PRT Artificial VII-19-B Hexapeptide 2 Arg Xaa Tyr Arg Trp Arg 1 53 6 PRT Artificial VII-23-B Hexapeptide 3 Arg Xaa Tyr Arg Trp Arg 1 5 46 PRT Artificial VII-31-B Hexapeptide 4 Arg Xaa Tyr Arg Trp Arg 1 5 5 6PRT Artificial VII-35-C Hexapeptide 5 Arg Xaa Tyr Arg Trp Arg 1 5 6 6PRT Artificial VII-43-C Hexapeptide 6 Arg Xaa Tyr Arg Trp Arg 1 5 7 6PRT Artificial VII-39-D Hexapeptide 7 Arg Xaa Tyr Arg Trp Arg 1 5 8 6PRT Artificial VII-51-A Hexapeptide 8 Arg Tyr Tyr Arg Xaa Arg 1 5 9 6PRT Artificial VII-53-B Hexapeptide 9 Xaa Tyr Tyr Arg Trp Arg 1 5 10 6PRT Artificial VII-55-A Hexapeptide 10 Arg Tyr Tyr Xaa Trp Arg 1 5 11 6PRT Artificial VII-57-C Hexapeptide 11 Arg Tyr Tyr Arg Trp Xaa 1 5 12 6PRT Artificial VII-61-B Hexapeptide 12 Arg Tyr Xaa Arg Trp Arg 1 5 13 6PRT Artificial VII-63-B Hexapeptide 13 Arg Tyr Xaa Arg Trp Arg 1 5 14 6PRT Artificial VII-65-F Hexapeptide 14 Xaa Tyr Tyr Arg Trp Arg 1 5 15 6PRT Artificial VII-73-A Hexapeptide 15 Arg Tyr Xaa Arg Trp Arg 1 5 16 6PRT Artificial VII-77-A Hexapeptide 16 Arg Tyr Xaa Arg Trp Arg 1 5 17 6PRT Artificial Second-position substitution. 17 Arg Xaa Tyr Arg Trp Arg1 5 18 6 PRT Artificial 3-position substitution. 18 Arg Tyr Xaa Arg TrpArg 1 5 19 6 PRT Artificial 1, 4, or 6 position substitution. 19 Xaa TyrTyr Xaa Trp Xaa 1 5 20 6 PRT Artificial VII-71-B Hexapeptide 20 Arg TyrXaa Arg Trp Arg 1 5 21 6 PRT Artificial VII-75-B Hexapeptide 21 Arg TyrXaa Arg Trp Arg 1 5 22 6 PRT Artificial VII-79-A Hexapeptide 22 Arg TyrXaa Arg Trp Arg 1 5 23 6 PRT Artificial VII-87-B Hexapeptide 23 Arg XaaTyr Arg Trp Arg 1 5 24 6 PRT Artificial IV-16-C Hexapeptide 24 Arg TyrTyr Arg Trp Arg 1 5 25 6 PRT Artificial IV-17-C Hexapeptide 25 Arg TyrTyr Arg Trp Arg 1 5 26 6 PRT Artificial VII-7-B Hexapeptide 26 Arg TyrTyr Arg Trp Arg 1 5 27 6 PRT Artificial 5-position substitution. 27 ArgTyr Tyr Arg Xaa Arg 1 5 28 6 PRT Artificial VII-1-A Hexapeptide 28 ArgTyr Tyr Arg Trp Arg 1 5 29 6 PRT Artificial VII-2-A hexapeptide. 29 ArgTyr Tyr Arg Trp Arg 1 5 30 6 PRT Artificial VII-3-A hexapeptide 30 ArgTyr Tyr Arg Trp Arg 1 5 31 6 PRT Artificial VII-4-B hexapeptide 31 ArgTyr Tyr Arg Trp Arg 1 5 32 6 PRT Artificial VII-11-B hexapeptide. 32 ArgXaa Xaa Arg Trp Arg 1 5 33 6 PRT Artificial VII-13-B hexapeptide. 33 ArgTyr Xaa Arg Trp Arg 1 5 34 6 PRT Artificial VII-17-B hexapeptide. 34 ArgXaa Xaa Arg Trp Arg 1 5 35 6 PRT Artificial VII-21-C hexapeptide. 35 ArgXaa Xaa Arg Trp Arg 1 5 36 6 PRT Artificial VII-27-B hexapeptide. 36 ArgXaa Tyr Arg Trp Arg 1 5 37 6 PRT Artificial VII-29-B hexapeptide. 37 ArgXaa Xaa Arg Trp Arg 1 5 38 6 PRT Artificial VII-33-B hexapeptide. 38 ArgXaa Xaa Arg Trp Arg 1 5 39 6 PRT Artificial VII-37-B hexapeptide. 39 ArgXaa Xaa Arg Trp Arg 1 5 40 6 PRT Artificial VII-49-B hexapeptide. 40 ArgXaa Xaa Arg Trp Arg 1 5 41 6 PRT Artificial VII-67-A hexapeptide. 41 XaaTyr Tyr Arg Trp Arg 1 5 42 6 PRT Artificial Base formula. 42 Arg Tyr TyrArg Trp Arg 1 5 43 6 PRT Artificial Base sequence with terminalmodifications. 43 Arg Tyr Tyr Arg Trp Arg 1 5 44 6 PRT ArtificialIV-12-B hexapeptide. 44 Arg Xaa Tyr Xaa Trp Arg 1 5 45 6 PRT ArtificialIV-15-A hexapeptide. 45 Arg Tyr Tyr Arg Trp Arg 1 5 46 6 PRT ArtificialIV-21-C hexapeptide. 46 Ala Tyr Tyr Arg Trp Arg 1 5 47 6 PRT ArtificialIV-23-B hexapeptide. 47 Arg Ala Tyr Arg Trp Arg 1 5 48 6 PRT ArtificialIV-25-B hexapeptide. 48 Arg Tyr Ala Arg Trp Arg 1 5 49 6 PRT ArtificialIV-27-B hexapeptide. 49 Arg Tyr Tyr Ala Trp Arg 1 5 50 6 PRT ArtificialIV-29-B hexapeptide. 50 Arg Tyr Tyr Arg Ala Arg 1 5 51 6 PRT ArtificialIV-31-B hexapeptide. 51 Arg Tyr Tyr Arg Trp Ala 1 5 52 6 PRT ArtificialIV-33-B hexapeptide. 52 Ala Tyr Tyr Arg Trp Arg 1 5 53 6 PRT ArtificialIV-35-B hexapeptide. 53 Arg Ala Tyr Arg Trp Arg 1 5 54 6 PRT ArtificialIV-37-B hexapeptide. 54 Arg Tyr Ala Arg Trp Arg 1 5 55 6 PRT ArtificialIV-39-B hexapeptide. 55 Arg Tyr Tyr Ala Trp Arg 1 5 56 6 PRT ArtificialIV-41-B hexapeptide. 56 Arg Tyr Tyr Arg Ala Arg 1 5 57 6 PRT ArtificialIV-43-B hexapeptide. 57 Arg Tyr Tyr Arg Trp Ala 1 5

What is claimed and desired to be secured by United States LettersPatent is:
 1. A hexapeptide of the formula: Arg-Xaa-Tyr-Arg-Trp-Arg (SEQID NO: 17), wherein the hexapeptide binds with the ORL-1 receptor,wherein xaa is selected from the group consisting of natural aminoacids, D-amino acids, non-natural amino acids, modified natural aminoacids, and amino acid analogs.
 2. The hexapeptide of claim 1, whereinXaa is a modified natural amino acid.
 3. The hexapeptide of claim 1,wherein Xaa is a modified Phe amino acid molecule.
 4. The hexapeptide ofclaim 3, wherein Xaa is an amino acid selected from the group consistingof Phe (4-Me), Phe (4-COOH), Phe (4-NO₂), Phe (4-F), Phe (4-CN).
 5. Thehexapeptide of claim 1, having the formula of SEQ ID NO:
 1. 6. Thehexapeptide of claim 1, having the formula of SEQ ID NO:
 2. 7. Thehexapeptide of claim 1, having the formula of SEQ ID NO:
 3. 8. Thehexapeptide of claim 1, having the formula of SEQ ID NO:
 4. 9. Thehexapeptide of claim 1, having the formula of SEQ ID NO:
 6. 10. Thehexapeptide of claim 1, wherein Xaa is a modified Tyr amino acidmolecule.
 11. The hexapeptide of claim 10, wherein Xaa is an amino acidmolecule selected from the group consisting of Tyr (4-Me), Tyr (3-Cl),and Tyr (BN, 3-Cl).
 12. The hexapeptide of claim 1, having the formulaof SEQ ID NO:
 5. 13. The hexapeptide of claim 1, having the formula ofSEQ ID NO:
 7. 14. The hexapeptide of claim 1, having the formula of SEQID NO:
 23. 15. The hexapeptide of claim 1, wherein the amino terminus ofthe peptide is acetylated.
 16. The hexapeptide of claim 1, wherein theamino terminus of the peptide is modified to include a moiety selectedfrom the group consisting of straight-chain alkyls, substitutedstraight-chain alkyls, branched-chain alkyls, substituted branched-chainalkyls, aralalkyls, cycloalkyls, or alkylcycloalkyls containing fromabout 1 to about 12 carbon atoms.
 17. The hexapeptide of claim 1,wherein the carboxy terminus is aminated.
 18. The hexapeptide of claim1, wherein the carboxy terminus is aminated and the amino terminus ismodified to include a moiety selected from the group consisting ofstraight-chain alkyls, substituted straight-chain alkyls, branched-chainalkyls, substituted branched chain alkyls, aralalkyls, cycloalkyls, oralkylcycloalkyls containing from about 1 to about 12 carbon atoms.
 19. Apharmaceutical composition comprising a hexapeptide of claim 4 and apharmaceutically acceptable diluent or excipient.
 20. A pharmaceuticalcomposition comprising a hexapeptide of claim 11 and a pharmaceuticallyacceptable diluent or excipient.
 21. A hexapeptide of the formula:Arg-Tyr-Xaa-Arg-Trp-Arg (SEQ ID NO: 18), wherein the hexapeptide bindswith the ORL-1 receptor, wherein Xaa is selected from the groupconsisting of natural amino acids, D-amino acids, non-natural aminoacids, modified natural amino acids, and amino acid analogs.
 22. Thehexapeptide of claim 21, wherein Xaa is a modified natural amino acid.23. The hexapeptide of claim 21, wherein Xaa is a modified Phe aminoacid molecule.
 24. The hexapeptide of claim 23, wherein Xaa is an aminoacid selected from the group consisting of Phe (4-F), Phe (NO₂), hPhe(2, 4 di-NO₂), Phe (4-CH₂SO₃H), Phe (4-NHAc), and Phe (4-CH₂NH₂). 25.The hexapeptide of claim 21, having the formula of SEQ ID NO:
 12. 26.The hexapeptide of claim 21, having the formula of SEQ ID NO:
 13. 27.The hexapeptide of claim 21, having the formula of SEQ ID NO:
 20. 28.The hexapeptide of claim 21, having the formula of SEQ ID NO:
 21. 29.The hexapeptide of claim 21, having the formula of SEQ ID NO:
 16. 30.The hexapeptide of claim 21, having the formula of SEQ ID NO:
 22. 31.The hexapeptide of claim 21, wherein Xaa is a modified Tyr amino acidmolecule.
 32. The hexapeptide of claim 31, wherein Xaa is Tyr (2, 6di-Me) (SEQ ID NO: 15).
 33. The hexapeptide of claim 21, wherein theamino terminus of the peptide is acetylated.
 34. The hexapeptide ofclaim 21, wherein the amino terminus of the peptide is modified toinclude a moiety selected from the group consisting of straight-chainalkyls, substituted straight-chain alkyls, branched-chain alkyls,substituted branched-chain alkyls, aralalkyls, cycloalkyls, oralkylcycloalkyls containing from about 1 to about 12 carbon atoms. 35.The hexapeptide of claim 21, wherein the carboxy terminus is aminated.36. The hexapeptide of claim 21, wherein the carboxy terminus isaminated and the amino terminus is modified to include a moiety selectedfrom the group consisting of straight-chain alkyls, substitutedstraight-chain alkyls, branched-chain alkyls, substituted branched-chainalkyls, aralalkyls, cycloalkyls, or alkylcycloalkyls containing fromabout 1 to about 12 carbon atoms.
 37. A pharmaceutical compositioncomprising a hexapeptide of claim 24 and a pharmaceutically acceptablediluent or excipient.
 38. A pharmaceutical composition comprising ahexapeptide of claim 32 and a pharmaceutically acceptable diluent orexcipient.
 39. A hexapeptide of the formula: Xaa₁-Tyr-Tyr-Xaa₂-Trp-Xaa₃(SEQ ID NO: 19), wherein the hexapeptide binds with the ORL-1 receptor,wherein Xaa is selected from the group consisting of natural aminoacids, D-amino acids, non-natural amino acids, modified natural aminoacids, and amino acid analogs.
 40. The hexapeptide of claim 39, whereinXaa is a modified natural amino acid.
 41. The hexapeptide of claim 39,wherein Xaa₁, Xaa₂, and Xaa₃ are selected from the group consisting ofArg, ε-aminocaproyl, and DAB.
 42. The hexapeptide of claim 39, havingthe formula of SEQ ID NO:
 9. 43. The hexapeptide of claim 39, having theformula of SEQ ID NO:
 10. 44. The hexapeptide of claim 39, having theformula of SEQ ID NO:
 11. 45. The hexapeptide of claim 39, having theformula of SEQ ID NO:
 14. 46. The hexapeptide of claim 39, having theformula of SEQ ID NO:
 24. 47. The hexapeptide of claim 39, wherein theamino terminus of the peptide is acetylated.
 48. The hexapeptide ofclaim 39, wherein the amino terminus of the peptide is modified toinclude a moiety selected from the group consisting of straight-chainalkyls, substituted straight-chain alkyls, branched-chain alkyls,substituted branched-chain alkyls, aralalkyls, cycloalkyls, oralkylcycloalkyls containing from about 1 to about 12 carbon atoms. 49.The hexapeptide of claim 39, wherein the carboxy terminus is aminated.50. The hexapeptide of claim 39, wherein the carboxy terminus isaminated and the amino terminus is modified to include a moiety selectedfrom the group consisting of straight-chain alkyls, substitutedstraight-chain alkyls, branched-chain alkyls, substituted branched-chainalkyls, aralalkyls, cycloalkyls, or alkylcycloalkyls containing fromabout 1 to about 12 carbon atoms.
 51. A pharmaceutical compositioncomprising a hexapeptide of claim 41 and a pharmaceutically acceptablediluent or excipient.
 52. A hexapeptide of the formula:Arg-Tyr-Tyr-Arg-Xaa-Arg (SEQ ID NO: 27), wherein the hexapeptide bindswith the ORL-1 receptor, wherein Xaa is selected from the groupconsisting of natural amino acids, D-amino acids, non-natural aminoacids, modified natural amino acids, and amino acid analogs.
 53. Thehexapeptide of claim 52, wherein Xaa is a modified natural amino acid.54. The hexapeptide of claim 52, wherein Xaa is a modified Trp aminoacid molecule.
 55. The hexapeptide of claim 54, wherein Xaa is Trp(5-CN) (SEQ ID NO: 8).
 56. The hexapeptide of claim 52, wherein theamino terminus of the peptide is acetylated.
 57. The hexapeptide ofclaim 52, wherein the amino terminus of the peptide is modified toinclude a moiety selected from the group consisting of straight-chainalkyls, substituted straight-chain alkyls, branched-chain alkyls,substituted branched-chain alkyls, aralalkyls, cycloalkyls, oralkylcycloalkyls containing from about 1 to about 12 carbon atoms. 58.The hexapeptide of claim 52, wherein the carboxy terminus is aminated.59. The hexapeptide of claim 52, wherein the carboxy terminus isaminated and the amino terminus is modified to include a moiety selectedfrom the group consisting of straight-chain alkyls, substitutedstraight-chain alkyls, branched-chain alkyls, substituted branched-chainalkyls, aralalkyls, cycloalkyls, or alkylcycloalkyls containing fromabout 1 to about 12 carbon atoms.
 60. A pharmaceutical compositioncomprising a hexapeptide of claim 55 and a pharmaceutically acceptablediluent or excipient.
 61. A method of treating pain comprisingadministering a compound comprising the peptide of claim 4 to a cell.62. A method of treating pain comprising administering a compoundcomprising the peptide of claim 11 to a cell.
 63. A method of treatingpain comprising administering a compound comprising the peptide of claim24 to a cell.
 64. A method of treating pain comprising administering acompound comprising the peptide of claim 32 to a cell.
 65. A method oftreating pain comprising administering a compound comprising the peptideof claim 41 to a cell.
 66. A method of treating pain comprisingadministering a compound comprising the peptide of claim 55 to a cell.